Myotonic dystrophy (DM) is caused by repeat nucleotide expansions, specifically CUG and CCUG expansions in the non-coding regions of the dystrophia myotonia protein kinase gene (DMPK) and the Zn finger 9 gene (ZNF9), respectively. Patients with the CUG expansions have type I myotonic dystrophy (DM1) and patients with the CCUG expansions have type II myotonic dystrophy (DM2). Patients with DM1 and DM2 display the same symptoms suggesting both CUG and CCUG expansions cause DM through a common mechanism. It is hypothesized that these non-coding expansions cause DM through an RNA gain-of-function mechanism; the expanded CUG and CCUG repeat RNAs act as toxic RNAs to sequester MBNL1, a RNA binding protein, and also indirectly increase the protein levels of CUGBP1, another RNA binding protein, which disrupts the normal cellular function of these proteins. MBNL1 and CUGBP1 are pre-mRNA splicing factors and decreasing and increasing their active concentration results in the mis- regulation of alternative splicing of multiple transcripts with an outcome of DM for those who harbor CUG and CCUG expansions. The expanded CUG repeats that cause DM1 are an excellent drug target because if one can identify a small molecule that binds CUG repeats, this binding event could release the sequestered MBNL1 and the expected outcome would be that MBNL1 would be able to properly regulate the splicing of its substrate pre-mRNAs and reduce or eliminate some of the symptoms of myotonic dystrophy. Towards this goal, we have identified a small molecule (pentamidine) that binds the CUG repeats and releases MBNL1, reverses the splicing defects associated with DM1 in a human DM1 cell model and partially reverses the splicing defects in a DM1 mouse model. Although pentamidine binds the CUG repeats and reverses some of the molecular defects associated with DM1, pentamidine appears to have limited specificity for the CUG repeats; therefore, analogues of pentamidine will be synthesized with the goal of obtaining small molecules with greater specificity and affinity for CUG repeats compared to pentamidine. The mechanism through which pentamidine disrupts the MNBL - CUG repeat interaction is being determined and this knowledge will be used to improve the design of analogues with improved activity. The long term goal of this work is to identify a compound that could lead to a therapy for patients with myotonic dystrophy type I for which no therapies are currently available. PUBLIC HEALTH RELEVANCE: The goal of this work is to identify compounds that could be used to develop therapies for myotonic dystrophy patients. Myotonic dystrophy is the second most common form of muscular dystrophy and is thought to affect in 1 in 8000 people.